Determination of vanilmandelic acid in urine

ABSTRACT

The concentration of vanilmandelic acid in urine is determined by contacting a measured urine sample with an anion exchange resin to adsorb contained vanilmandelic acid, washing residual urine from the resin, desorbing vanilmandelic acid from the resin, diazotizing the desorbed vanilmandelic acid, extracting the diazotized vanilmandelic acid from the diazotization reaction mixture with cyclohexanone, determining the absorbance of about 590 Mu light by the extract and determining vanilmandelic acid content of the extract by comparing the absorbance of the extract with the absorbance of solutions having known contents of vanilmandelic acid.

Tlnited tates tent [191 James Sept. 17, T974 DETERMINATION OFVANILMANDELIIC ACTD TN URINE [75] Inventor: Gerald E. James, San Mateo,Calif.

[7 3] Assignee: Oxford Laboratories, Foster City,

Calif.

[22] Filed: June 6, 1973 [21] Appl. No.: 367,632

[52] US. Cl. 23/230 B, 252/408 [51] lint. Cl ..G01n 21/20, GOln 33/16[58] Field of Search 23/230 B [56] References Cited UNITED STATESPATENTS 3,482,942 l2/l969 Schneider 23/230 B OTHER PUBLICATIONS StanleyE. Gitlow et al., Am. J. Cardiology, 26(3), 2709(1970). ClinicalDiagnosis, I. Davidson et al., eds., 14th Edition, 622-624, W. B.Saunders, Phila., 1969.

Gradwohls Clinical Laboratory Methods, S. Frankel et al., eds., Vol. I,279, 282-3, C. V. Mosby, St. Louis, 1970.

Primary Examiner-Morris O. Wolk Assistant Examiner-Sidney Marantz [5 7ABSTRACT The concentration of vanilmandelic acid in urine is determinedby contacting a measured urine sample with an anion exchange resin toadsorb contained vanilmandelic acid, washing residual urine from theresin, desorbing vanilmandelic acid from the resin, diazotizing thedesorbed vanilmandelic acid, extracting the diazotized vanilmandelicacid from the diazotization reaction mixture with cyclohexanone,determining the absorbance of about 590 ,1, light by the extract anddetermining vanilmandelic acid content of the extract by comparing theabsorbance of the extract with the absorbance of solutions having knowncontents of vanilmandelic acid.

3 Claims, No Drawings BACKGROUND OF THE INVENTION In 1957 Armstrong etal reported that vanilmandelic acid (3-methoxy-4-hydroxymandelic acid)is a major urinary metabolite of the catecholamines and noted that theurinary excretion of vanilmandelic acid is increased in cases ofpheochromocytoma. (Armstrong et a1 3-methoxy-4-hydroxy-D-mandelic acid,and urinary metabolite of norepinephrine. Biochim. Biophys. Acta 25,422-423 (1957)). Since. this report it has been generally accepted thatabnormally high contents of vanilmandelic acid in urine were indicativeof pheochromocytoma. Accurate determination of vanilmandelic acid inurine is a difficult undertaking. The problem has been studiedextensively and a number of methods for making the determination havebeen reported. (Vanilmandelic Acid Determinations Second Edition, Beeleret a1, Commission on Continuing Education Council on Clinical ChemistryCopyright 1968 American Society of Clinical Pathologists).

In 1960 Gitlow et al described a colorimetric urine test forpheochromocytoma. (Gitlow et a1 American Journal of Medicine, June 1960,P. 921). Gitlow et a1 extracted vanilmandelic acid from urine,diazotized the extract, extracted the diazotized vanilmandelic acid withnormal amyl alcohol containing a minor amount of ethanolamine anddetermined the absorbance of 450 and 550 mu light by the extract andthen determined the vanilmandelic acid content of the sample bycomparison with the absorbance of solutions having known contents ofvanilmandelic acid which had been subjected to the same procedure. Sincethe Gitlow et al publication several variations of their method havebeen published. All'of the methods involve preparing a concentrate ofvanilmandelic acid from a urine sample, diazotizing the vanilmandelicacid contained in the concentrate, extracting the diazotizedvanilmandelic acid with an organic solvent and determining theabsorbance of the extract with varying wavelengths of light and thencomparing this absorbance with that of solutions having knownvanilmandelic acid contents which had been put through the sameprocedure.

BRIEF DESCRIPTION OF THE INVENTION Methods of determining vanilmandelicacid in urine by preparing a vanilmandelic acid concentrate from theurine, adding a diazotizing agent to the concentrate, extracting theresultant mixture with an organic solvent and determining the opticaldensity of the extract are improved by using cyclohexanone as theorganic solvent and determining optical density with light having awavelength about 590 millimicrons. For example, urine is added to acolumn of anion exchange resin under conditions such that thevanilmandelic acid is held by the resin while amines and acids weakerthan vanilmandelic acid are washed off. Then vanilmandelic acid plussome other phenolic acids are eluted with sodium chloride. The eluate isbuffered to pH 10.0 and the vanilmandelic acid is diazotized withpnitrodiazonium chloride. The diazo compound is extracted intocyclohexanone and the organic layer is separated and washed with aqueouscarbonate to ensure that the diazo compound is in its deep blue, basicform. The absorbance of the organic layer is measured at 590 mu. Theextraction eliminates most of the inter- LII ferences remaining afterthe column chromatography or renders their absorbance almost negligibleat 590 nm.

DETAILED DESCRIPTION OF THE INVENTION REAGENTS l. Acetic Acid, 1M

2. Sodium Chloride, 3.9M

3. Potassium Carbonate/Bicarbonate, 4M, pH 11.5

4. Cyclohexanone 5. p-Nitroaniline in aqueous ll-ICl (p-Nitroaniline1.66g/L, I-lCl 55 ml conc. HCl/L) 6. Solid Sodium Nitrite 7.vanilmandelic Acid Standard, 50 mg/L (keep refrigerated) 8. ResinColumns REAGENT PREPARATION Dissolve the sodium nitrite in deionizedwater and dilute to ml (keep refrigerated).

SAMPLE PREPARATION The preferred sample is a 24-hour urine preservedwith 10 ml of concentrated HCl. The pH of the urine should be adjustedto between 4 and 8 before proceeding. COLUMN CHROMATOGRAPHY PROCEDUREColumns with the funnels attached are needed for two standards, 5 mg/Land 10 mg/L, and a blank, in addition to one per sample.

1. Add 10 ml deionized water to each column.

2. Add the following:

To the blank column nothing.

To the 5 mg/L standard column 100 microliters vanilmandelic acidstandard.

To the 10 mg/L standard column 200 microliters vanilmandelic acidstandard.

to each sample column 1 ml urine.

3. After a couple of minutes (time not critical) cut the tips off thecolumns and allow them to drain (to waste).

4. Add 12 ml acetic acid (IN) to each column and drain (to waste).

5. Add 10 ml deionized water to each column and drain (to waste).

6. Repeat Step 5.

7. Position a test tube which will contain at least 20 ml under eachcolumn.

8. Add 10 ml of sodium chloride solution (3.9M) to each column and alloweach column to drain into test tubes.

COLORIMETRY PROCEDURE 9. Add 1 ml potassium carbonate/bicarbonate buffer(pH 11.5-11.8) to each tube and mix well.

10. Prepare fresh diazo reagent by mixing:

One volume p-nitroaniline with two volumes sodium nitrite solution (2g/L).

For each test tube 0.2 ml of diazo reagent will be needed. Use withinten minutes.

11. Add 200 microliters of the fresh diazo reagent to each tube and mixwell as each addition is made.

12. Wait five minutes and add 5 ml cyclohexanone to each tube and mixthe two phases vigorously.

13. Allow the two phases to separate (2-3 minutes),

then remove the lower (aqueous) layer.

14. Add 2 ml of the potassium carbonate/bicarbonate buffer (pH11.5-11.8) to each tube and mix the two phases vigorously.

l5. Centrifuge at 1,500 rpm for about minutes to separate the phases andensure a clear upper phase.

16. Transfer the upper phase to a cuvette which requires about four mlto cover the photometer light path. Take care not to disturb the lowerphase.

17. Read the absorbance of the upper phase for each standard and samplevs. the blank at 590 mu. (Hitachi l24,u.v Visible Double Beamspectrophotometer or Bausch & Lomb spectronic 88 are suitable).

CALCULATIONS Plot absorbance (O.D.) vs. concentration in mg/L for thestandards.

Read concentrations of samples in mg/L from the graph.

To report vanilmandelic in mg/24 hours:

mg/L X 24-hour urine volume in liters hours.

NOTES:

1. The washing of the resin in steps 4 and 5 may be done with largevolumes of deionized water but the use of a dilute solution of a weakorganic acid is preferred, such washing permits quicker and morecomplete removal of residual urine.

2. The use of the buffer solution in steps 9 and 14 is convenient butadjustment of pH may be made with any alkaline solution.

3. The blank" at the end of the procedure is generally yellow in color,diazotized vanilmandelic acid in cyclohexanone is blue, vanilmandelicacid concentrations below about 8mg/L appear greenish. Highervanilmandelic acid concentrations show a definite blue color.

4. The selection of the anion exchange resin is not critical; anystrongly basic resin will suffice. Suitable resins include the Type Iresins made by Dow Chemical Co., and sold under the trade name Dowex andthose made by Diamond Shamrock Co., and sold under the trade nameDuolile.

The method for determining vanilmandelic acid described above is fast,accurate and reproducible. The cyclohexanone extract of the diazotizedvanilmandelic acid is blue in color. Any high reading of vanilmandelicacid not attended by definite blue color in the cyclohexanone extract issuspect and should be checked.

Light at a wavelength about 590 millimicrons gives superior absorbancespectra with cyclohexanone extracts. The method eliminates mostinterferences remaining after the adsorption step or renders theirabsorbance negligible at 590 millimicrons.

I claim:

1. In a method for determining the content of vanilmandelic acid inurine by preparing a concentrate of vanilmandelic acid from the urine,diazotizing the vanilmandelic acid contained in the concentrate,extracting the diazotized vanilmandelic acid with an organic solvent anddetermining the optical density of the extract, the improvement whichcomprises using cyclohexanone as the organic solvent.

2. The method as defined in claim 1 wherein the optical density isdetermined using light having a wavelength about 590 millimicrons.

3. The method of determining the quantity of vanilmandelic acid in urinewhich comprises:

a. contacting a measured sample of urine having its pH adjusted to alevel in the range about 4 to 8 with an anion exchange resin to adsorbcontained vanilmandelic acid,

b. washing the resin to remove residual urine,

0. contacting the resin with aqueous sodium chloride to desorbvanilmandelic acid and recovering aqueous sodium chloride containingdesorbed vanilmandelic acid,

d. adjusting the pH of the recovered aqueous sodium chloride to a levelin the range 9-1 1,

e. mixing freshly prepared p-nitrodiazonium chloride with the recoveredaqueous sodium chloride,

f. extracting the mixture obtained in (e) with cyclohexanone,

g. intimately mixing the cyclohexanone extract with dilute aqueousalkali and recovering the cyclohexanone phase from the mixture,

h. determining the optical density of the cyclohexanone phase at about590 mg,

i. determining the vanilmandelic acid content of the sample bycomparison with the optical density of solutions having known contentsof vanilmandelic acid in the range about 5-15 mg/L which have beensubjected to steps a-h inclusive.

2. The method as defined in claim 1 wherein the optical density isdetermined using light having a wavelength about 590 millimicrons. 3.The method of determining the quantity of vanilmandelic acid in urinewhich comprises: a. contacting a measured sample of urine having its pHadjusted to a level in the range about 4 to 8 with an anion exchangeresin to adsorb contained vanilmandelic acid, b. washing the resin toremove residual urine, c. contacting the resin with aqueous sodiumchloride to desorb vanilmandelic acid and recovering aqueous sodiumchloride containing desorbed vanilmandelic acid, d. adjusting the pH ofthe recovered aqueous sodium chloride to a level in the range 9-11, e.mixing freshly prepared p-nitrodiazonium chloride with the recoveredaqueous sodium chloride, f. extracting the mixture obtained in (e) withcyclohexanone, g. intimately mixing the cyclohexanone extract withdilute aqueous alkali and recovering the cyclohexanone phase from themixture, h. determining the optical density of the cyclohexanone phaseat about 590 m Mu , i. determining the vanilmandelic acid content of thesample by comparison with the optical density of solutions having knowncontents of vanilmandelic aCid in the range about 5-15 mg/L which havebeen subjected to steps a-h inclusive.